Pancreatolithiasis and pancreatic carcinoma. Evaluation of pancreatic excretion test with 5,5-dimethyl-2,4-oxazolidinedione.

The pancreatic excretion test with a weak acid of 5, 5-dimethyl-2,4-oxazolidinedione (DMO) was performed concomitantly with the pancreozymin-secretin test in 28 patients with pancreatolithiasis, 14 patients with pancreatic carcinoma, and 67 healthy subjects. The DMO concentration and total output of duodenal content after secretin stimulation, when corrected to the simultaneously determined plasma DMO concentration, were significantly reduced in the patients. While the pancreozymin-secretin test was abnormal in 96% of patients with pancreatolithiasis and in 86% of those with pancreatic carcinoma, the pancreatic DMO excretion test gave abnormal results in 100% of the patients. This suggests that the new test may well become effective in detecting early stages of pancreatic disease including carcinoma and chronic pancreatitis.

Trimethadione metabolism, a useful indicator for assessing hepatic drug-oxidizing capacity.

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1, 1A2, 2A1, 2A2, 2B1, 2B2, 2C6, 2C7, 2C11, 2C12, 2C13, 2E1, 3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2, 2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Induction of cytochrome P-450 and related drug-oxidizing activities in muscone (3-methylcyclopentadecanone)-treated rats.

In the present study, we investigated the effects of muscone on both in vitro and in vivo parameters of the hepatic microsomal drug-metabolizing enzyme system and other enzyme activities in rats. In the in vivo study, the serum dimethadione (DMO)/trimethadione (TMO) ratios at 2 hr after oral administration of TMO (100 mg/kg) were significantly increased in both male and female rats treated with 75 and 150 but not 40 mg muscone/kg. Antipyrine metabolite profile in 24 hr urine of rats pretreated with muscone (150 mg/kg) was examined. The results showed that the excretion of norantipyrine was significantly increased as compared to the control group. In the in vitro study, we found that the content of cytochrome P-450, and activities of aminopyrine, N-demethylase, aniline hydroxylase and delta-aminolevulinic acid (ALA) synthetase were significantly increased as compared to the controls in both male and female rats treated with muscone (75 and 150 mg/kg). This type of induction of the hepatic metabolizing enzymes was similar to that seen after treatment with a prototype drug, phenobarbital.

Species differences in pharmacokinetics and drug teratogenesis.

Interspecies differences in regard to the teratogenicity of drugs can be the result of differing pharmacokinetic processes that determine the crucial concentration-time relationships in the embryo. Maternal absorption, as well as distribution, of the drugs does not usually show great species differences. The first-pass effect after oral application is often more pronounced in animals than man (e.g., valproic acid, 13-cis-retinoic acid), although in some cases the reverse was found (e.g., hydrolysis of valpromide). Existing differences can be adjusted by appropriate choice of the administration route and measurements of drug levels. Many variables determine the placental transfer of drugs: developmental stage, type of placenta, properties of the drug. Even closely related drugs (e.g., retinoids) may differ greatly in regard to placental transfer. Maternal protein binding is an important determinant of placental transfer, since only the free concentration in maternal plasma can equilibrate with the embryo during organogenesis; this parameter differs greatly across species (e.g., valproic acid: five times higher free fractions in mouse and hamster than in monkey and man). The metabolic pattern has not yet been demonstrated to be a major cause of species differences, although recent evidence on phenytoin and thalidomide support the hypothesis that some species differences can be the result of differing activation/deactivation pathways. Laboratory animals usually have a much higher rate of drug elimination than man. Drastic drug level fluctuations are therefore present during teratogenicity testing in animals, but not to the same degree in human therapy. It must, therefore, be investigated if peak concentrations (such as for valproic acid and possibly caffeine) or the area under the concentration-time curve (AUC) (such as for cyclophosphamide and possibly retinoids) correlate with the teratogenic response. Only then is a rational and scientific basis for interspecies comparison possible. It is concluded that the prediction of the human response based on animal studies can be improved by consideration of the appropriate pharmacokinetic determinants.

Comparability of in vitro and in vivo methods for the determination of alterations in drug metabolism.

The above series of experiments taken in toto suggest the usefulness of lindane as a model substrate for studying the effects of a variety of compounds on drug metabolism in vivo. Excellent correlations were observed in comparison with the in vitro measurements both qualitatively and quantitatively. Unlike some of the other compounds discussed lindane offers some distinct advantages. One is that because the metabolites can be monitored in the urine, it is non-invasive in nature. A second is that a number of mixed function oxidase pathways (phase I reactions) can be determined at the same time. This would be of great importance if the effect of a compound is rather selective and does not alter the single pathway measured in the metabolism of other substrates which have been suggested as model compounds. However, the tradeoff is obviously the need for more analytical work. A third advantage is the ability of the system to detect changes in conjugative or phase II reactions at the same time. Further studies will be necessary with all of these model substrates to detect their usefulness and their limitations.

Dose-independent pharmacokinetics of trimethadione and its metabolite in rats.

The aim of the present study was to examine the dose-independent kinetics of trimethadione (1) and its only metabolite dimethadione, 5,5-dimethyl-2,4-oxazolidinedione (2), after oral administration of 1-, 2-, and 4-mg/kg doses of 1 to rats. Pharmacokinetic parameters determined after oral administration of these doses showed that the half-life (t 1/2), metabolic clearance (CL), and apparent volume of distribution (Vd) were not significantly changed by increasing or decreasing the dose of 1, whereas there was a linear relationship between the dose of 1 and the area under the curve (AUC) (1, r = 0.912; 2, r = 0.976) or the maximum serum concentration (Cmax) (1, r = 0.990; 2, r = 0.980). The ratios of 2 to 1 at 1 and 2 h after oral administration of 1 were not significantly different. These experiments indicate that serum pharmacokinetic behavior of 1 and 2 1 or 2 h after oral administration of 1 to the rat is independent of the dose of 1 in the 1-4 mg/kg range.

Trimethadione metabolism and microsomal monooxygenases in untreated and phenobarbital-treated rhesus monkeys.

The contribution of induced cytochrome P450 (P450) isozymes (CMLa; CYP2B, CMLb; CYP2A and CMLc; CYP3A) and related enzymes to trimethadione (TMO) metabolism in phenobarbital-treated rhesus monkey were investigated. The animals received a single dose of TMO (4 mg kg-1) and plasma samples were withdrawn before this administration and again at 0.08, 0.25, 0.5, 1 and 2 h later. Phenobarbital-treatment (20 mg kg-1 day-1 for 3 days; i.p.) significantly increased the plasma dimethadione (DMO)/TMO ratios at 0.08, 0.5, 1 and 2 h one's appropriate controls. Phenobarbital treatment also increased the P450 content (1.7-fold) and activity of aniline p-hydroxylase (1.3-fold), p-nitroanisole O-demethylase (1.8-fold) and benzphetamine N-demethylase (2.3-fold). The content of CMLa, CMLb and CMLc were increased about 12.8, 2.3 and 2.7-fold by phenobarbital pretreatment, respectively. The activity of TMO N-demethylation was inhibited by anti-P450 CMLa and anti-P450 CMLb. However, the anti-P450 CMLc antibody had no effect on this activity in liver microsomes. The results of both in vivo and in vitro studies of the effects of phenobarbital treatment on TMO metabolism indicate that these effects may be attributed to the induction of CMLa. These findings suggest that plasma DMO/TMO ratio in a single blood sampling after TMO administration is very useful for determination the degree of hepatic induction in clinical study.

[Measurement of microsomal function in hepatectomized rat].

Trimethadione (TMO) is exclusively metabolized to dimethadione (DMO) by cytochrome P-450 dependent mono-oxygenase in liver microsomes. We have chosen TMO as an indicator compound for estimating the microsomal function of the liver. TMO tolerance test was performed in the CCl4 intoxicated injury or 68% partial-hepatectomized rats. Serum DMO/TMO ratios in 2 hours following oral administration of TMO in these rats in vivo were well correlated with changes in content of hepatic cytochrome P-450 and activity of TMO N-demethylase in vitro. TMO tolerance test could also determine quantitatively the changes of the functional capacity of the liver in the 68% partial hepatectomy. These results suggest that TMO tolerance test is a useful tool for the surgical indication in the hepatectomy.

Trimethadione metabolism as a probe drug to estimate hepatic oxidizing capacity in rats.

1. Trimethadione (TMO) has the properties required of probe drugs for the evaluation of hepatic oxidizing capacity in vivo. 2. TMO is demethylated to dimethadione (DMO), its only metabolite, in the liver after oral administration. 3. In rats with various types of hepatic intoxicated-, induced- and partially hepatectomized-rats, the serum DMO/TMO ratios, which were measured on blood samples obtained by a single collection 2 hr after oral administration of TMO, correlated well with the degree of hepatic damage or induction. 4. This finding suggests that TMO may be used as a probe drug in the rapid determination of the functional reserve mass of the liver as well as the hepatic oxidizing capacity.

A method for estimation of hepatic drug-metabolizing capacity: determination of concentration of trimethadione and its metabolite in human serum.

There have been significant correlations between serum concentration ratios of 5,5-dimethyl-2,4-oxazolidinedione (Dimethadione, DMO)/trimethadione (TMO) after administration of TMO and hepatic microsomal cytochrome P-450 contents in rats with various treatments (CCl4 or phenobarbital). The pharmacokinetics of TMO and DMO, and the serum concentration ratio of DMO/TMO have been investigated in healthy volunteers after oral administration of 1 mg/kg (N = 4), 2 mg/kg (N = 6) and 4 mg/kg (N = 6) TMO, respectively. TMO and DMO concentrations in serum were determined by a gas-liquid chromatographic method. Serum disappearance of TMO was described by one compartment model. The T1/2, Kel, Vd and C1 of TMO and of DMO were shown to have almost the same values in 2 mg/kg or 4 mg/kg TMO administration. Correlation coefficient between DMO/TMO ratio in serum and time course after 1 mg/kg, 2 mg/kg and 4 mg/kg TMO administration was found to be r = 0.958, r = 0.924 and r = 0.938, respectively. These results indicate that the serum concentration ratio of DMO/TMO, especially at 2 or 4 h after 4 mg/kg TMO administration orally, may be an index of hepatic drug-metabolizing capacity in human serum as well as in rats.

Trimethadione tolerance test for evaluation of functional reserve of the liver in patients with liver cirrhosis and esophageal varices.

Dimethadione (DMO)/trimethadione (TMO) ratios in serum after oral administration of TMO were investigated in 15 normal subjects and in 20 patients with cirrhosis and esophageal varices. Severe impairment of liver function was associated with a decrease in total cholesterol, total protein and plasma albumin, or an increase in total bilirubin, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, serum gamma-glutamyl transpeptidase and indocyanine green retention rate (ICG R15). Serum concentration ratios of DMO to TMO at 2 or 4 h after oral administration of TMO in patients were significantly decreased by 67 or 66%, respectively, compared to normal subjects. DMO/TMO ratios at 2 or 4 h following oral administration of TMO were well correlated with the liver function parameters (plasma albumin r = 0.758 at 2 h, r = 0.776 at 4 h; total protein r = 0.613 at 2 h, r = 0.619 at 4 h; ICG R15 r = -0.683 at 2 h, r = -0.746 at 4 h, in patients only; cholinesterase r = 0.873 at 2 h, r = 0.908 at 4 h) as well as with pharmacokinetic parameters (total body clearance r = 0.794 at 2 h, r = 0.786 at 4 h) in both the normal subjects and the patients. It suggests that the DMO/TMO ratio obtained in a single blood sample collected after oral administration of TMO might provide a useful measure for function hepatic reserve.

Comparative pharmacokinetic study of trimethadione and dimethadione for lysis of pancreatic stones.

In order to investigate the biological equivalence of dimethadione (DMO) and its precursor trimethadione (TMO), comparative pharmacokinetic studies were performed in 6 beagle dogs. DMO and TMO power was given orally at the same molar dose of 1.0 g and 1.11 g (7.7 mM), respectively. Plasma concentration of DMO and TMO was determined at an appropriate interval. Four pharmacokinetic parameters were calculated from the plasma concentration-time curves of DMO and TMO; the peak plasma concentration (Cmax), the time to the peak concentration of Cmax (Tmax), the area under the plasma concentration-time curve (AUC), and the biological half-life (t 1/2). In the plasma concentration-time curve of DMO, Tmax after oral administration of TMO was longer than that after administration of DMO. This resulted from the presence of the conversion process of TMO to DMO in the body, because of the very short Tmax observed in the plasma concentration-time curve of TMO. Cmax, AUC and t 1/2 did not differ significantly between the drugs. The results obtained indicate that TMO is biologically equivalent to DMO in regard to plasma concentration of DMO.

Trimethadione N-demethylation by rat liver CYP2E1 in vitro.

Trimethadione (TMO) is a model drug utilized for estimation of hepatic metabolism in clinical studies, and it was reported that TMO N-demethylase activity was inhibited by CYP2E1 inhibitors and substrates in rat in vivo. This study was performed to investigate the involvement of the CYP2E1 subfamily on TMO N-demethylation in vitro and to clarify these inhibitory mechanisms. The effects of acetone (AC), imidazole (IM) and N-nitrosodimethylamine (NDA) on TMO N-demethylation were studied in vitro. Rat hepatic microsomal fractions were employed as the enzyme source of TMO N-demethylase and the activity was determined by the production of dimethadione (DMO). DMO was analyzed by a GC/FTD equipped with a narrow-bore capillary column. TMO N-demethylation was biphasic by the graphic analysis of Eadie-Hofstee plots; this suggests the involvement of at least two enzymes in TMO metabolism in the rat. The kinetic parameters for the formation of DMO were analyzed graphically using double-reciprocal plots. The apparent K(m1), K(m2) and Vmax1, Vmax2 values for DMO formation were 4, 20 mM and 182, 595 pmol/mg protein/min, respectively. AC and IM inhibited TMO N-demethylase activity competetively. However, mixed inhibition kinetics was observed by NDA. Furthermore, TMO N-demethylase activity was inhibited by antiserum to CYP2E1 by 62% and CYP3A2 by 46%. These results indicate that the CYP2E1 subfamily is the major enzyme involved in TMO N-demethylation in rat in vitro although the CYP3A2 is also involved in this transformation.

Effect of dimethadione derived from repeated oral administration of trimethadione on pancreatic secretion in dogs.

The effect of the weak organic acid of dimethadione (DMO) on secretin-stimulated pancreatic secretion was studied with repeated oral administration of trimethadione (TMO), the precursor of DMO, to dogs at a dose of 10 to 160mg/kg/day for a period of 14 days. The bicarbonate concentration in pancreatic juice at a steady state decreased significantly, reflecting a close correlation with the dose of TMO and DMO concentrations in plasma and pancreatic juice. The maximal decrement from the control of cases of no TMO administration was 18.8 mEq/l (12.1% of the control level). The chloride concentration in pancreatic juice showed a reciprocal relation to the bicarbonate concentration. The sum of both anion concentration was constant, irrespective of the dose of TMO. The average carbon dioxide tension of pancreatic juice in all doses of TMO was lower than that of the control, but differences were not statistically significant. The pH, flow rate, sodium and potassium concentrations in pancreatic juice at a steady state did not differ significantly in relation to the dose of TMO. These findings suggest that repeated oral administration of TMO cause a significant decrease in bicarbonate concentration in pancreatic juice, resulting probably from the buffer action of bicarbonate on protons provided from the undissociated form of DMO.

Pancreatic excretion of dimethadione and trimethadione by repeated oral administration of trimethadione in dogs.

To examine pancreatic excretion of dimethadione (DMO), a weak organic acid, as well as of its precursor trimethadione (TMO), TMO was given orally to dogs with pancreatic fistulae at a dose of 10-160 mg/kg/day over a period of 14 days. Blood samples were taken once a day during the administration of TMO and for 7 days after discontinuation of the drug. On the 15th day, pancreatic juice was collected under stimulation by secretin (2 Crick-Haper-Raper units/kg/h). DMO concentration in plasma reached a maximal plateau around the 10th day after starting TMO administration, and depended directly on the dose of TMO. Pancreatic excretion of DMO at a steady state closely depended on both the dose of TMO and the DMO concentration in plasma. The pancreatic juice/plasma concentration ratio for DMO exceeded 1.0 at a steady rate and decreased with the increased flow rate. Pancreatic DMO clearance (DMO output/DMO concentration in plasma) increased, depending on the flow rate, the bicarbonate concentration, and pH of pancreatic juice. Pancreatic excretion of TMO was zero or extremely low.

Saliva levels of trimethadione and its metabolite as an index of drug metabolizing activity in rats.

1. It was investigated in normal and liver-injured rats administered with trimethadione (TMO) as to whether concentrations of TMO in plasma are able to be predicted from the corresponding saliva levels. 2. The saliva/plasma (S/P)ratio of TMO was about 1, while its metabolite 5,5-dimethyl-2,4-oxazolidinedione (DMO) showed a S/P ratio of 0.65. 3. In normal rats, there was a high correlation between saliva and plasma concentration of TMO and DMO. (TMO: r=0.966, DMO: r=0.950). 4. In liver-injured pretreated rats with carbon tetrachloride, alpha-naphthyl isothiocyanate or D-galactosamine, there was a high correlation between the saliva DMO/TMO ratio and the plasma DMO/TMO ratio after oral administration of TMO (r=0.983 at 1 h, r=0.952 at 2 h). 5. The saliva DMO/TMO ratio, as well as the plasma DMO/TMO ratio, may be utilized as an index of drug-metabolizing activity of the liver.

Effects of cimetidine and diethylaminoethyl 2,2-diphenylvalerate HCL (SKF 525-A) on trimethadione metabolism in the rat.

The effects of cimetidine and diethylaminoethyl 2,2-diphenylvalerate (SKF 525-A) on trimethadione (TMO) metabolism were examined in the rat. The pretreatment of rats with cimetidine (100 mg/kg, i.p.) or SKF 525-A (40 mg/kg, i.p.) resulted in a prolongation of half-life (T1/2), an increase in the area under the curve (AUC) and a decrease in the total body clearance (CL) of TMO. However, the apparent volume of distribution (Vd) of TMO was not changed. The activities of hepatic cytochrome P-450-dependent drug-oxidizing enzymes such as aminopyrine and TMO N-demethylase, and aniline hydroxylase activities were decreased by pretreatment of rats with cimetidine or SKF 525-A. Cytochrome P-450 contents were not changed. The inhibition of aminopyrine and TMO N-demethylase activities in the rat pretreated with cimetidine or SKF 525-A was noncompetitive. These results suggest that cimetidine and SKF 525-A inhibited TMO metabolism in a similar manner to the rat pretreated with their doses.

Effects of cimetidine and ranitidine on trimethadione metabolism in the rat.

Pretreatment of rats with cimetidine (100 mg/kg, i.p.) resulted in a prolongation of TMO half-life, an increase in the area under the curve (AUC) and a decrease of clearance (Cl), whereas in the rats pretreated with ranitidine (120 mg/kg, i.p.), these parameters were not changed. The apparent volume of distribution (Vd) values were not changed by either of the drugs as compared to controls. Activities of hepatic cytochrome P-450-dependent metabolizing enzymes such as aminopyrine- and TMO N-demethylase and aniline hydroxylase activity were decreased by pretreatment of rats with cimetidine, whereas in the rats pretreated with ranitidine, these enzyme activities were not changed. Cytochrome P-450 contents were not changed by either of the drugs as compared to controls. The inhibition manner of aminopyrine- and TMO N-demethylase activities in the rats pretreated with cimetidine was noncompetitive. These results indicate, together with the previous findings, that cimetidine treatment inhibited TMO metabolism, but ranitidine did not.